I. Tocopherol Synthesis Pathway
The chloroplast of higher plants exhibit interconnected biochemical pathways that lead to secondary metabolites, including tocopherols, that not only perform functions in plants but can also be important for mammalian nutrition. In plastids, tocopherols account up to 40% of the total quinone pool. The biosynthetic pathway of α-tocopherol in higher plants involves condensation of homogentisic acid and phytylpyrophosphate to form 2-methyl-6 phytylbenzoquinol (Fiedler et al., Planta 155: 511-515 (1982); Soll et al., Arch. Biochem. Biophys. 204: 544-550 (1980); Marshall et al., Phytochem. 24: 1705-1711 (1985), all of which are herein incorporated by reference in their entirety). The plant tocopherol biosynthetic pathway can be divided into four parts: synthesis of homogentisic acid, which contributes to the aromatic ring of tocopherol; synthesis of phytylpyrophosphate, which contributes to the side chain of tocopherol; cyclization which plays a role in chirality and chromanol substructure of the vitamin E family; and S-adenosyl methionine dependent methylation of an aromatic ring, which effects the compositional quality of the vitamin E family.
Homogentisate is an aromatic precursor in the biosynthesis of tocopherols in chloroplasts and is formed from the aromatic shikimate metabolite, p-hydroxyphenylpyruvate. The aromatic amino acids phenylalanine, tyrosine, and tryptophan are formed by a reaction sequence that initiates from the two carbohydrate precursors, D-erythrose 4-phosphate and phosphoenolpyruvate, via shikimate, and forms prearomatic and aromatic compounds (Bentley, Critical Rev. Biochem. Mol. Biol. 25: 307-384 (1990), the entirety of which is herein incorporated by reference). Approximately 20% of the total carbon fixed by green plants is routed through the shikimate pathway with end products being aromatic amino acids and other aromatic secondary metabolites such as flavonoids, vitamins, lignins, alkaloids, and phenolics (Herrmann, Plant Physiol. 107: 7-12 (1995), Kishore and Shah, Ann. Rev. Biochem. 57:67-663 (1988), both of which are herein incorporated by reference in their entirety). Various aspects of the shikimate pathway have been reviewed (Bentley, Critical Rev. Biochem. Mol. Biol. 25: 307-384 (1990); Herrmann, Plant Physiol. 107: 7-12 (1995); Kishore and Shah, Ann. Rev. Biochem. 57:67-663 (1988)).
The first reported committed reaction in the shikimate pathway is catalyzed by the enzyme 3-deoxyarabino-heptulosonate 7-phosphate synthase (also known as 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, deoxyarabino-heptulosonate-P-synthase, and DAHP synthase (EC. 4.1.2.15)), which has been reported to control carbon flow into the shikimate pathway. The plastid localized DAHP synthase catalyzes the formation of 3-deoxy-D-arabino-heptulosonate 7-phosphate by condensing D-erythrose 4-phosphate with phosphoenolpyruvate. DAHP synthase has been isolated from plant sources including carrot and potato. DAHP synthase has substrate specificity for D-erythrose 4-phosphate and phosphoenolpyruvate, is a dimer of subunits having a molecular weight of 53 KD and is activated by Mn2+ (Herrmann, Plant Physiol. 107: 7-12 (1995)). Aromatic amino acids are not reported to act as feedback regulators. Purified DAHP synthase is activated by tryptophan and, to a lesser extent, by tyrosine in a hysteric fashion (Suzich et al., Plant Physiol. 79: 765-770 (1985), the entirety of which is herein incorporated by reference).
The next reported enzyme in the shikimate pathway is 3-dehydroquinate synthase (EC 4.6.1.3), which catalyzes the formation of dehydroquinate, the first carbocyclic metabolite in the biosynthesis of aromatic amino acids, from the substrates D-erythrose 4-phosphate and phosphoenolpyruvate. The enzyme reaction involves a NAD (nicotinamide adenine dinucleotide) cofactor dependent oxidation-reduction, β-elimination, and an intramolecular aldol condensation. 3-dehydroquinate synthase has been purified from Phaseolus mungo seedlings and pea seedlings, has a native molecular weight of 66 KD and is a dimer (Yamamoto, Phytochem. 19: 779-802 (1980); Pompliano et al., J. Am. Chem. Soc. 111: 1866-1871-1871 (1989), both of which are herein incorporated by reference in their entirety).
3-dehydroquinate dehydratase (EC 4.2.1.10) catalyzes the stereospecific syn-dehydration of dehydroquinate to dehydroshikimate and has been reported to be responsible for initiating the process of aromatization by introducing the first of three double bonds of the aromatic ring system. 3-dehydroquinate dehydratase has been cloned from E. coli (Duncan et al., Biochem. J. 238:475-483 (1986), the entirety of which is herein incorporated by reference).
Shikimate dehydrogenase (EC 1.1.1.25) catalyzes the NADPH (reduced nicotinamide adenine dinucleotide phosphate)-dependent conversion of dehydroshikimate to shikimate. Bifunctional 3-dehydroquinate dehydratase-shikimate dehydrogenase has been reported in spinach, pea seedling, and Maize (Bentley, Critical Rev. Biochem. Mol. Biol. 25: 307-384 (1990), Kishore and Shah, Ann. Rev. Biochem. 57:67-663 (1988)). E. coli shikimate dehydrogenase has been reported to be a monomeric, monofunctional protein with a molecular weight of 32,000 daltons (Chaudhuri and Coggins, Biochem. J. 226:217-223 (1985), the entirety of which is herein incorporated by reference).
Shikimate kinase (EC 2.7.1.71) catalyzes the phosphorylation of shikimate to shikimate-3-phosphate. Shikimate kinase exists as isoforms in E. coli and S. typhimurium. Plant shikimate kinase has been partially purified from mung bean and sorghum (Bentley, Critical Rev. Biochem. Mol. Biol. 25: 307-384 (1990); Kishore and Shah, Ann. Rev. Biochem. 57:67-663 (1988)). Certain plant species accumulate shikimate and shikimate kinase may play a role in regulating flux in the tocopherol synthesis pathway.
5-Enolpyruvyl-shikimate-3-phosphate synthase (also known as enolpyruvyl-shikimate-P-synthase, and EPSPS (EC 2.5.1.19)) catalyzes the reversible transfer of the carboxyvinyl moiety of phosphoenolpyruvate to shikimate-3-phosphate, yielding 5-enolpyruvyl-shikimate-3-phosphate. 5-Enolpyruvyl-shikimate-3-phosphate synthase is a target of the broad spectrum, nonselective, postemergence herbicide, glyphosate. Chemical modification studies indicate that lysine, arginine, and histidine residues are essential for activity of the enzyme (Kishore and Shah, Ann. Rev. Biochem. 57:67-663 (1988)). 5-Enolpyruvyl-shikimate-3-phosphate synthase has been isolated and characterized from microbial and plant sources including tomato, petunia, Arabidopsis, and Brassica (Kishore and Shah, Ann. Rev. Biochem, 57:67-663 (1988)).
Chorismate synthase (EC 4.6.1.4) catalyzes the conversion of 5-enolpyruvyl-shikimate-3-phosphate to chorismic acid and introduces a second double bond in an aromatic ring and a trans-1,4-elimination of inorganic phosphorous. Chorismate is the last reported common intermediate in the biosynthesis of aromatic compounds via the shikimate pathway. The enzyme reaction involves no change in the oxidation state of the substrate. Chorismate synthase from various sources requires a reduced flavin cofactor, FMNH2 (reduced flavin mononucleotide) or FADH2 (reduced flavin adenine dinucleotide), for catalytic activity (Bentley, Critical Rev. Biochem. Mol. Biol. 25: 307-384 (1990); Kishore and Shah, Ann. Rev. Biochem. 57:67-663 (1988)).
The next reported enzyme in the tocopherol biosynthetic pathway is chorismate mutase (EC 5.4.99.5), which catalyzes the conversion of chorismic acid to prephenic acid. Chorismic acid is a substrate for a number of enzymes involved in the biosynthesis of aromatic compounds. Plant chorismate mutase exists in two isoforms, chorismate mutase-1 and chorismate mutase-2, that differ in feedback regulation by aromatic amino acids (Singh et al., Arch. Biochem. Biophys. 243: 374-384 (1985); Goers et al., Planta 162: 109-124 (1984), both of which are herein incorporated by reference in their entirety). It has been reported that chloroplastic chorismate mutase-1 may play a role in biosynthesis of aromatic amino acids as this enzyme is activated by tyrosine and phyenlalanine. Cytosolic isozyme chorismate mutase-2 is not regulated by aromatic amino acids and may play a role in providing the aromatic nucleus for synthesis of aromatic secondary metabolites including tocopherol (d'Amato et al., Planta, 162: 104-108 (1984), the entirety of which is herein incorporated by reference).
The metabolic pathways branch after prephenic acid and lead not only to phenylalanine and tyrosine, but also to a number of secondary metabolites. Tyrosine is synthesized from prephenate via either 4-hydroxyphenylpyruvate or arogenate. Both routes have been reported in plants (Bentley, Critical Rev. Biochem. Mol. Biol. 25: 307-384 (1990)).
The formation of 4-hydroxyphenylpyruvate from prephenate is catalyzed by prephenate dehydrogenase (EC 1.3.1.12 for NAD specific prephenate dehydrogenase and EC 1.3.1.13 for NADP specific prephenate dehydrogenase). 4-Hydroxyphenylpyruvate associated with tocopherol biosynthesis may also come from tyrosine pool by the action of tyrosine transaminase (EC 2.6.1.5) or L-amino acid oxidase (EC 1.4.3.2). Tyrosine transaminase catalyzes the pyridoxal-phosphate dependent conversion of L-tyrosine to 4-hydroxyphenylpyruvate. This reversible enzyme reaction transfers the amino group of tyrosine to 2-oxoglutarate to form 4-hydroxyphenylpyruvate and glutamate. L-amino acid oxidase (EC 1.4.3.2) catalyzes the conversion of tyrosine to 4-hydroxyphenylpyruvate by acting on the amino group of tyrosine with oxygen acting as an acceptor. L-amino acid oxidase is not specific to tyrosine. In E. coli, aromatic amino acid amino transferase (also referred to as aromatic-amino-acid transaminase (EC 2.6.1.57)) converts 4-hydroxyphenylpyruvate to tyrosine and plays a role in phenylalanine and tyrosine biosynthesis (Oue et al., J. Biochem. (Tokyo) 121: 161-171 (1997); Soto-Urzua et al., Can. J. Microbiol. 42: 294-298 (1996); Hayashi et al., Biochemistry 32: 12229-12239 (1993), all of which are herein incorporated by reference in their entirety).
Aspartic acid amino transferase or transaminase A (EC 2.6.1.1) exhibits a broad substrate specificity and may utilize phenylpyruvate or p-hydroxyphenylpyruvate to form phenylalanine and tyrosine, respectively. Transaminase A has been characterized in Aradidopsis (Wilkie et al., Biochem J. 319: 969-976 (1996); Wilkie et al., Plant Mol. Biol. 27: 1227-1233 (1995), both of which are herein incorporated by reference in their entirety), rice (Song et al., DNA Res. 3: 303-310 (1996), herein incorporated by reference in its entirety), Panicum miliaceum L (Taniguchi et al., Arch. Biochem. Biophys. 318: 295-306 (1995), herein incorporated by reference in its entirety), Lupinus angustifolius (Winefield et al., Plant Physiol. 104: 417-423 (1994), herein incorporated by reference in its entirety), and soybean (Wadsworth et al., Plant Mol. Biol. 21: 993-1009 (1993), herein incorporated by reference in its entirety).
A precursor molecule, homogentisic acid, is produced in the chloroplast from the shikimate pathway intermediate p-hydroxyphenylpyruvate. p-Hydroxyphenylpyruvate dioxygenase (also known as 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27)) catalyzes the formation of homogentisate from hydroxyphenylpyruvate through an oxidative decarboxylation of the 2-oxoacid side chain accompanied by hydroxylation of the aromatic ring and a 1,2 migration of the carboxymethyl group. Norris et al. reported functional identification of a pdsI gene that encodes p-Hydroxyphenylpyruvate dioxygenase (Norris et al., Plant Cell 7: 2139-2149 (1995), the entirety of which is herein incorporated by reference). p-Hydroxyphenylpyruvate dioxygenase has been cloned from Arabidopsis and carrot (GENBANK accession numbers U89267, AF000228, and U87257; Garcia et al., Biochem. J. 325: 761-769 (1997), herein incorporated by reference in its entirety). Fiedler et al. reported the localization and presence of this enzyme in both isolated spinach chloroplast and the peroxisome (Fiedler et al., Planta, 155: 511-515 (1982)). Garcia et al. reported the purification of the cytosolic form of hydroxyphenylpyruvate dioxygenase from cultured carrot protoplast (Garcia et al., Biochem. J. 325: 761-769 (1997), the entirety of which is herein incorporated by reference). It has been reported that the chloroplastic isoform may be involved in the biosynthesis of prenylquinones, and that the peroxisomal and cytosolic isoform may be involved in the degradation of tyrosine.
The carbon flow to the pool of phytol, i.e., the isoprene-derived side chain of tocopherol, occurs via the mevalonate pathway or non-mevalonate pathway. Geranylgeranyl-pyrophosphate synthase (GGPP synthase (EC 2.5.1.29)) catalyzes the formation of geranylgeranylpyrophosphate by prenyltransferring an isoprene moiety from isopentenylpyrophosphate to farnesylpyrophosphate. A gene encoding geranylgeranyl-pyrophosphate synthase has been isolated from Arabidopsis and Cantharanthus roseus (Zhu et al., Plant Cell Physiol. 38: 357-361 (1997), Bantignies et al., Plant Physiol. 110: 336-336 (1995), both of which are herein incorporated by reference in their entirety). Geranylgeranylpyrophosphate synthesized by GGPP synthase is used in the carotenoid and tocopherol biosynthesis pathways.
The NADPH-dependent hydrogenation of geranylgeranylpyrophosphate is catalyzed by geranylgeranylpyrophosphate hydrogenase (also called geranylgeranylpyrophosphate reductase) to form phytylpyrophosphate (Soll et al., Plant Physiol. 71: 849-854 (1983), the entirety of which is herein incorporated by reference). Geranylgeranylpyrophosphate hydrogenase appears to be localized to two sites: the chloroplast envelope and the thylakoids. The chloroplast envelope form is reported to be responsible for the hydrogenation of geranylgeranylpyrophosphate to a phytyl moiety. The thylakoids form is reported to be responsible for the stepwise reduction of chlorophyll esterified with geranylgeraniol to chlorophyll esterified with phytol. The chloroplast envelope form of geranylgeranylpyrophosphate may play a role in tocopherol and phylloquinone synthesis. A chlP gene cloned from Synechocystis has been functionally assigned by complementation in Rhodobactor sphaeroids to catalyze the stepwise hydrogenation of geranylgeraniol to phytol (Addlesse et al., FEBS Lett. 389: 126-130 (1996), the entirety of which is herein incorporated by reference).
Homogentisate:phytyl transferase (also referred to as phytyl/prenyltransferase) catalyzes the decarboxylation followed by condensation of homogentisic acid with a phytol moiety from phytylpyrophosphate to form 2-methyl-6 phytylbenzoquinol. Prenyltransferase activity has been reported in spinach chloroplasts and such activity is located in chloroplast envelope membranes (Fiedler et al., Planta 155: 511-515 (1982)). A reported prenyltransferase gene, termed pdsII, specific to tocopherol biosynthesis has been identified in Arabidopsis (Norris et al., Plant Cell 7: 2139-2149 (1995)).
Tocopherol cyclase catalyzes the cyclization of 2,3-dimethyl-6-phytylbenzoquinol to form γ-tocopherol and plays a role in the biosynthesis of enantioselective chromanol substructure of the vitamin E subfamily (Stocker et al., Bioorg. Medic. Chem. 4: 1129-1134 (1996), the entirety of which is herein incorporated by reference). The preferred substrate specificity of tocopherol cyclase may be either 2,3-dimethyl-6-phytylbenzoquinol or 2-methyl-5-phytylbenzoquinol or both. The substrate, 2-methyl-6 phytylbenzoquinol, is formed by prenyltransferase and requires methylation by an S-adenosylmethionine-dependent methyltransferase before cyclization. Tocopherol cyclase has been purified from green algae chlorella protothecoids, Dunaliella salina and from wheat leafs (U.S. Pat. No. 5,432,069, the entirety of which is herein incorporated by reference.
Synthesis of γ-tocopherol from 2-methyl-6 phytylbenzoquinol occurs by two pathways with either δ-tocopherol or 2,3 dimethyl-5-phytylbenzoquinol acting as an intermediate. α-Tocopherol is then synthesized from γ-tocopherol in a final methylation step with S-adenosylmethionine. These steps of α-tocopherol biosynthesis are located in the chloroplast membrane in higher plants. Formation of α-tocopherol from other tocopherols is catalyzed by S-adenosyl methionine (SAM)-dependent γ-tocopherol methyltransferase (EC 2.1.1.95). This enzyme has been partially purified from Capsicum and Euglena gracilis (Shigeoka et al., Biochim. Biophys. Acta 1128: 220-226 (1992), d'Harlingue and Camara, J. Biol. Chem. 260: 15200-15203 (1985), both of which are herein incorporated by reference in their entirety).
Tocotrienols are similar to tocopherols in molecular structure except that there are three double bonds in the isoprenoid side chain. Although tocotrienols have not been reported in soybean, they are found within in the plant kingdom. The tocotrienol biosynthetic pathway is similar to that of tocopherol up to the formation of homogentisic. It has been reported that homgentisate:phytyl transferase is able to transfer geranylgeranyl-pyrophosphate (“GGPP”) to homogentisic acid. A side chain of GGPP may be desaturated by the addition of phytylpyrophosphate to homogentisate. Stocker et al. report that a reduction of the side chain's double bond occurs at an earlier stage of the biosynthesis. Phytylpyrophosphate or GGPP are condensed with homogentisic acid (“HGA”) to yield different hydroquinone precursors which are cyclyzed by the same enzyme (Stocker et al., Bioorg. Medicinal Chem. 4:1129-1134 (1996), the entirety of which is herein incorporated by reference).
The primary oxidation product of tocopherol is tocopheryl quinone, which can be conjugated to yield glucuronate after prior reduction to the hydroquinone. In animals, glucuronate can be excreted into bile or further catabolized to tocopheronic acid in the kidney and processed for urinary excretion (Traber and Sies, Ann. Rev. Nutr. 16:321-347 (1996), the entirety of which is herein incorporated by reference).
In Aspergillus nidulans, the aromatic amino acid catabolic pathway involves formation of homogentisic acid followed by aromatic ring cleavage by an homogentisic acid dioxygenase (EC 1.13.11.5) to yield, after an isomerization step, fumarylacetoacetate (Fernandez-Canon et al., Anal. Biochem. 245: 218-22 (1997); Hudecova et al., Int. J. Biochem. Cell Biol. 27: 1357-1363 (1995); Fernandez-Canon et al., J. Biol. Chem. 270: 21199-21205 (1995), all of which are herein incorporated by reference in their entirety). Fumarylacetoacetate, is then split by fumarylacetoacetate (Fernandez-Canon and Penalva, J. Biol. Chem. 270:21199-21205 (1995), the entirety of which is herein incorporated by reference). Homogentisic acid dioxygenase uses a tocopherol biosynthetic metabolite homogentisic acid for hydrolysis.
Tocopherol levels are reported to vary in different plants, tissues, and developmental stages. The production of homogentisic acid by p-hydroxyphenylpyruvate dioxygenase may be a regulatory point for bulk flow through the pathway due to the irreversible nature of the enzyme reaction and due to the fact that homogentisic acid production is the first committed step in tocopherol biosynthesis (Norris et al., Plant Cell 7: 2139-2149 (1995)). Another regulatory step in tocopherol biosynthesis may be associated with the availability of phytylpyrophosphate pool. Feeding studies in Safflower callus culture showed 1.8-fold and 18-fold increase in tocopherol synthesis by feeding homogentisate and phytol, respectively (Fury et al., Phytochem. 26: 2741-2747 (1987), the entirety of which is herein incorporated by reference). In meadow rescue leaf, vitamin E increases in the initial phase of senescence when phytol is cleaved off from the chlorophylls and when a free phytol pool is available (Peskier et al., J. Plant Physiol. 135: 428-432 (1989), the entirety of which is herein incorporated by reference).
II. Expressed Sequence Tag Nucleic Acid Molecules
Expressed sequence tags, or ESTs are randomly sequenced members of a cDNA library (or complementary DNA)(McCombie et al., Nature Genetics 1:124-130 (1992); Kurata et al., Nature Genetics 8: 365-372 (1994); Okubo, et al. Nature Genetics 2: 173-179 (1992), all of which references are incorporated herein in their entirety). The randomly selected clones comprise insets that can represent a copy of up to the full length of a mRNA transcript.
Using conventional methodologies, cDNA libraries can be constructed from the mRNA (messenger RNA) of a given tissue or organism using poly dT primers and reverse transcriptase (Efstratiadis et al. Cell 7:279-288 (1976), the entirety of which is herein incorporated by reference; Higuchi et al., Proc. Natl. Acad. Sci. (U.S.A.) 73:3146-3150 (1976), the entirety of which is herein incorporated by reference; Maniatis et al., Cell 8:163-182 (1976) the entirety of which is herein incorporated by reference; Land et al., Nucleic Acids Res. 9:2251-2266 (1981), the entirety of which is herein incorporated by reference; Okayama et al., Mol. Cell. Biol. 2:161-170 (1982), the entirety of which is herein incorporated by reference; Gubler et al., Gene 25:263-269 (1983), the entirety of which is herein incorporated by reference).
Several methods may be employed to obtain full-length cDNA constructs. For example, terminal transferase can be used to add homopolymeric tails of dC residues to the free 3′ hydroxyl groups (Land et al., Nucleic Acids Res. 9:2251-2266 (1981), the entirety of which is herein incorporated by reference). This tail can then be hybridized by a poly dG oligo which can act as a primer for the synthesis of full length second strand cDNA. Okayama and Berg, Mol. Cell. Biol. 2: 161-170 (1982), the entirety of which is herein incorporated by reference, report a method for obtaining full length cDNA constructs. This method has been simplified by using synthetic primer-adapters that have both homopolymeric tails for priming the synthesis of the first and second strands and restriction sites for cloning into plasmids (Coleclough et al., Gene 34:305-314 (1985), the entirety of which is herein incorporated by reference) and bacteriophage vectors (Krawinkel et al., Nucleic Acids Res. 14:1913 (1986), the entirety of which is herein incorporated by reference; Han et al., Nucleic Acids Res. 15:6304 (1987), the entirety of which is herein incorporated by reference).
These strategies have been coupled with additional strategies for isolating rare mRNA populations. For example, a typical mammalian cell contains between 10,000 and 30,000 different mRNA sequences (Davidson, Gene Activity in Early Development, 2nd ed., Academic Press, New York (1976). The number of clones required to achieve a given probability that a low-abundance mRNA will be present in a cDNA library is N=(ln(1−P))/(ln(1−1/n)) where N is the number of clones required, P is the probability desired, and 1/n is the fractional proportion of the total mRNA that is represented by a single rare mRNA (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989), the entirety of which is herein incorporated by reference).
A method to enrich preparations of mRNA for sequences of interest is to fractionate by size. One such method is to fractionate by electrophoresis through an agarose gel (Pennica et al., Nature 301:214-221 (1983), the entirety of which is herein incorporated by reference). Another such method employs sucrose gradient centrifugation in the presence of an agent, such as methylmercuric hydroxide, that denatures secondary structure in RNA (Schweinfest et al., Proc. Natl. Acad. Sci. (U.S.A.) 79:4997-5000 (1982), the entirety of which is herein incorporated by reference).
A frequently adopted method is to construct equalized or normalized cDNA libraries (Ko, Nucleic Acids Res. 18:5705-5711 (1990), the entirety of which is herein incorporated by reference; Patanjali, S. R. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1943-1947 (1991), the entirety of which is herein incorporated by reference). Typically, the cDNA population is normalized by subtractive hybridization (Schmid et al., J. Neurochem. 48:307-312 (1987) the entirety of which is herein incorporated by reference; Fargnoli et al., Anal. Biochem. 187:364-373 (1990) the entirety of which is herein incorporated by reference; Travis et al., Proc. Natl. Acad. Sci (U.S.A.) 85:1696-1700 (1988) the entirety of which is herein incorporated by reference; Kato, Eur. J. Neurosci. 2:704-711 (1990); and Schweinfest et al., Genet. Anal. Tech. Appl. 7:64-70 (1990), the entirety of which is herein incorporated by reference). Subtraction represents another method for reducing the population of certain sequences in the cDNA library (Swaroop et al., Nucleic Acids Res. 19:1954 (1991), the entirety of which is herein incorporated by reference).
ESTs can be sequenced by a number of methods. Two basic methods may be used for DNA sequencing, the chain termination method of Sanger et al., Proc. Natl. Acad. Sci. (U.S.A.) 74: 5463-5467 (1977), the entirety of which is herein incorporated by reference, and the chemical degradation method of Maxam and Gilbert, Proc. Nat. Acad. Sci. (U.S.A.) 74: 560-564 (1977), the entirety of which is herein incorporated by reference. Automation and advances in technology such as the replacement of radioisotopes with fluorescence-based sequencing have reduced the effort required to sequence DNA (Craxton, Methods 2: 20-26 (1991), the entirety of which is herein incorporated by reference; Ju et al., Proc. Natl. Acad. Sci. (U.S.A.) 92: 4347-4351 (1995), the entirety of which is herein incorporated by reference; Tabor and Richardson, Proc. Natl. Acad. Sci. (U.S.A.) 92: 6339-6343 (1995), the entirety of which is herein incorporated by reference). Automated sequencers are available from, for example, Pharmacia Biotech, Inc., Piscataway, N.J. (Pharmacia ALF), LI-COR, Inc., Lincoln, Nebr. (LI-COR 4,000) and Millipore, Bedford, Mass. (Millipore BaseStation).
In addition, advances in capillary gel electrophoresis have also reduced the effort required to sequence DNA and such advances provide a rapid high resolution approach for sequencing DNA samples (Swerdlow and Gesteland, Nucleic Acids Res. 18:1415-1419 (1990); Smith, Nature 349:812-813 (1991); Luckey et al., Methods Enzymol. 218:154-172 (1993); Lu et al., J. Chromatog. A. 680:497-501 (1994); Carson et al., Anal. Chem. 65:3219-3226 (1993); Huang et al., Anal. Chem 64:2149-2154 (1992); Kheterpal et al., Electrophoresis 17:1852-1859 (1996); Quesada and Zhang, Electrophoresis 17:1841-1851 (1996); Baba, Yakugaku Zasshi 117:265-281 (1997), all of which are herein incorporated by reference in their entirety).
ESTs longer than 150 nucleotides have been found to be useful for similarity searches and mapping (Adams et al., Science 252:1651-1656 (1991), herein incorporated by reference). ESTs, which can represent copies of up to the full length transcript, may be partially or completely sequenced. Between 150-450 nucleotides of sequence information is usually generated as this is length of sequence information that is routinely and reliably produced using single run sequence data. Typically, only single run sequence data is obtained from the cDNA library (Adams et al., Science 252:1651-1656 (1991). Automated single run sequencing typically results in an approximately 2-3% error or base ambiguity rate (Boguski et al., Nature Genetics 4:332-333 (1993), the entirety of which is herein incorporated by reference).
EST databases have been constructed or partially constructed from, for example, C. elegans (McCombrie et al., Nature Genetics 1:124-131 (1992)), human liver cell line HepG2 (Okubo et al., Nature Genetics 2:173-179 (1992)), human brain RNA (Adams et al., Science 252:1651-1656 (1991)); Adams et al., Nature 355:632-635 (1992)), Arabidopsis, (Newman et al., Plant Physiol. 106:1241-1255 (1994)); and rice (Kurata et al., Nature Genetics 8:365-372 (1994)).
III. Sequence Comparisons
A characteristic feature of a protein or DNA sequence is that it can be compared with other known protein or DNA sequences. Sequence comparisons can be undertaken by determining the similarity of the test or query sequence with sequences in publicly available or proprietary databases (“similarity analysis”) or by searching for certain motifs (“intrinsic sequence analysis”)(e.g. cis elements) (Coulson, Trends in Biotechnology 12: 76-80 (1994), the entirety of which is herein incorporated by reference); Birren et al., Genome Analysis 1: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 543-559 (1997), the entirety of which is herein incorporated by reference).
Similarity analysis includes database search and alignment. Examples of public databases include the DNA Database of Japan (DDBJ)(available on the Worldwide Web at ddbj.nig.ac.jp/); Genebank (available on the Worldwide Web at ncbi.nlm.nih.gov/Web/Search/Index.html); and the European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL) (available on the Worldwide Web at ebi.ac.uk/ebi_docs/embl_db/embl-db.html). A number of different search algorithms have been developed, one example of which are the suite of programs referred to as BLAST programs. There are five implementations of BLAST, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology 12: 76-80 (1994); Birren et al., Genome Analysis 1: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 543-559 (1997)).
BLASTN takes a nucleotide sequence (the query sequence) and its reverse complement and searches them against a nucleotide sequence database. BLASTN was designed for speed, not maximum sensitivity, and may not find distantly related coding sequences. BLASTX takes a nucleotide sequence, translates it in three forward reading frames and three reverse complement reading frames, and then compares the six translations against a protein sequence database. BLASTX is useful for sensitive analysis of preliminary (single-pass) sequence data and is tolerant of sequencing errors (Gish and States, Nature Genetics 3: 266-272 (1993), the entirety of which is herein incorporated by reference). BLASTN and BLASTX may be used in concert for analyzing EST data (Coulson, Trends in Biotechnology 12: 76-80 (1994); Birren et al., Genome Analysis 1: 543-559 (1997)).
Given a coding nucleotide sequence and the protein it encodes, it is often preferable to use the protein as the query sequence to search a database because of the greatly increased sensitivity to detect more subtle relationships. This is due to the larger alphabet of proteins (20 amino acids) compared with the alphabet of nucleic acid sequences (4 bases), where it is far easier to obtain a match by chance. In addition, with nucleotide alignments, only a match (positive score) or a mismatch (negative score) is obtained, but with proteins, the presence of conservative amino acid substitutions can be taken into account. Here, a mismatch may yield a positive score if the non-identical residue has physical/chemical properties similar to the one it replaced. Various scoring matrices are used to supply the substitution scores of all possible amino acid pairs. A general purpose scoring system is the BLOSUM62 matrix (Henikoff and Henikoff, Proteins 17: 49-61 (1993), the entirety of which is herein incorporated by reference), which is currently the default choice for BLAST programs. BLOSUM62 is tailored for alignments of moderately diverged sequences and thus may not yield the best results under all conditions. Altschul, J. Mol. Biol. 36: 290-300 (1993), the entirety of which is herein incorporated by reference, describes a combination of three matrices to cover all contingencies. This may improve sensitivity, but at the expense of slower searches. In practice, a single BLOSUM62 matrix is often used but others (PAM40 and PAM250) may be attempted when additional analysis is necessary. Low PAM matrices are directed at detecting very strong but localized sequence similarities, whereas high PAM matrices are directed at detecting long but weak alignments between very distantly related sequences.
Homologues in other organisms are available that can be used for comparative sequence analysis. Multiple alignments are performed to study similarities and differences in a group of related sequences. CLUSTAL W is a multiple sequence alignment package that performs progressive multiple sequence alignments based on the method of Feng and Doolittle, J. Mol. Evol. 25: 351-360 (1987), the entirety of which is herein incorporated by reference. Each pair of sequences is aligned and the distance between each pair is calculated; from this distance matrix, a guide tree is calculated, and all of the sequences are progressively aligned based on this tree. A feature of the program is its sensitivity to the effect of gaps on the alignment; gap penalties are varied to encourage the insertion of gaps in probable loop regions instead of in the middle of structured regions. Users can specify gap penalties, choose between a number of scoring matrices, or supply their own scoring matrix for both pairwise alignments and multiple alignments. CLUSTAL W for UNIX and VMS systems is available at: ftp.ebi.ac.uk. Another program is MACAW (Schuler et al., Proteins Struct. Func. Genet. 9:180-190 (1991), the entirety of which is herein incorporated by reference, for which both Macintosh and Microsoft Windows versions are available. MACAW uses a graphical interface, provides a choice of several alignment algorithms, and is available by anonymous ftp at: ncbi.nlm.nih.gov (directory/pub/macaw).
Sequence motifs are derived from multiple alignments and can be used to examine individual sequences or an entire database for subtle patterns. With motifs, it is sometimes possible to detect distant relationships that may not be demonstrable based on comparisons of primary sequences alone. Currently, the largest collection of sequence motifs in the world is PROSITE (Bairoch and Bucher, Nucleic Acid Research 22: 3583-3589 (1994), the entirety of which is herein incorporated by reference). PROSITE may be accessed via either the ExPASy server on the World Wide Web or anonymous ftp site. Many commercial sequence analysis packages also provide search programs that use PROSITE data.
A resource for searching protein motifs is the BLOCKS E-mail server developed by S. Henikoff, Trends Biochem Sci. 18:267-268 (1993), the entirety of which is herein incorporated by reference; Henikoff and Henikoff, Nucleic Acid Research 19:6565-6572 (1991), the entirety of which is herein incorporated by reference; Henikoff and Henikoff, Proteins, 17: 49-61 (1993). BLOCKS searches a protein or nucleotide sequence against a database of protein motifs or “blocks.” Blocks are defined as short, ungapped multiple alignments that represent highly conserved protein patterns. The blocks themselves are derived from entries in PROSITE as well as other sources. Either a protein query or a nucleotide query can be submitted to the BLOCKS server; if a nucleotide sequence is submitted, the sequence is translated in all six reading frames and motifs are sought for these conceptual translations. Once the search is completed, the server will return a ranked list of significant matches, along with an alignment of the query sequence to the matched BLOCKS entries.
Conserved protein domains can be represented by two-dimensional matrices, which measure either the frequency or probability of the occurrences of each amino acid residue and deletions or insertions in each position of the domain. This type of model, when used to search against protein databases, is sensitive and usually yields more accurate results than simple motif searches. Two popular implementations of this approach are profile searches (such as GCG program ProfileSearch) and Hidden Markov Models (HMMs) (Krough. et al., J. Mol. Biol. 235:1501-1531, (1994); Eddy, Current Opinion in Structural Biology, 6:361-365, (1996), both of which are herein incorporated by reference in their entirety). In both cases, a large number of common protein domains have been converted into profiles, as present in the PROSITE library, or HHM models, as in the Pfam protein domain library (Sonnhammer et al., Proteins 28:405-420 (1997), the entirety of which is herein incorporated by reference). Pfam contains more than 500 HMM models for enzymes, transcription factors, signal transduction molecules, and structural proteins. Protein databases can be queried with these profiles or HMM models, which will identify proteins containing the domain of interest. For example, HMMSW or HMMFS, two programs in a public domain package called HMMER (Sonnhammer et al., Proteins 28:405-420, (1997)) can be used.
PROSITE and BLOCKS represent collected families of protein motifs. Thus, searching these databases entails submitting a single sequence to determine whether or not that sequence is similar to the members of an established family. Programs working in the opposite direction compare a collection of sequences with individual entries in the protein databases. An example of such a program is the Motif Search Tool, or MoST (Tatusov et al. Proc. Natl. Acad. Sci. 91: 12091-12095 (1994), the entirety of which is herein incorporated by reference). On the basis of an aligned set of input sequences, a weight matrix is calculated by using one of four methods (selected by the user). A weight matrix is simply a representation, position by position of how likely a particular amino acid will appear. The calculated weight matrix is then used to search the databases. To increase sensitivity, newly found sequences are added to the original data set, the weight matrix is recalculated, and the search is performed again. This procedure continues until no new sequences are found.